ADAMTS nucleic acids and proteins

ABSTRACT

The present invention is directed to ADAMTS3 and the corresponding protein ADAMTS-3 as well as variants, homologs, and equivalents, and their use in procollagen processing.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/296,384, filed Jun. 6, 2001.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to isolated nucleic acidmolecules encoding proteins, and the proteins themselves, belonging to asubfamily of zinc metalloproteases referred to as “ADAMTS”, anabbreviation for A Disintegrin-like And Metalloprotease domain withThromboSpondin type I motifs. Proteins in the ADAMTS subfamily allpossess a Zn protease catalytic site consensus sequence (HEXXH+H), whichsuggests an intact catalytic activity for each of these proteins. TheADAMTS proteins also have putative N-terminal signal peptides and lacktransmembrane domains, which suggests that the proteins in thissubfamily are secreted. The proteins in the ADAMTS subfamily alsopossess at least one thrombospondin type (TSP 1) motif, which suggests abinding of these proteins to components of the extracellular matrix(ECM) or to cell surface components.

[0003] U.S. Pat. No. 6,391,610, which is incorporated herein in itsentirety by reference thereto, describes certain members of the ADAMTSfamily member and their implications in a variety of diseases. It isbelieved that members of the ADAMTS family play a role in the cleavageof proteoglycan core proteins found in the extracellular matrix (e.g.versican, brevican, neuracan, and aggrecan, as well as collagen). It isexpected that other members of the ADAMTS subfamily play a role inembryogenesis, implantation of a fertilized egg, angiogenesis, arthriticdegradation of cartilage, inflammation, nerve regeneration, tumorgrowth, and metastases.

[0004] Collagens comprise the major structural proteins of theextracellular matrix (ECM) and exist in both fibril-forms (e.g.,collagens I, II, III, V and XI) and nonfibrillar forms. Moleculesbelonging to both categories are homo-trimeric (e.g., collagen II) orhetero-trimeric (e.g., collagen I) assemblies of specific a-chains, eachthe product of a single gene. The molecular forms of collagens, as wellas the specific supramolecular aggregates and assemblies they form, areoften tissue specific, and provide specialized functions. For example,collagen I, the principal collagen of skin, is arranged in randomlyoriented bundles which form a sheet in the dennis, but is arranged inparallel bundles in tendons. This reflects the different mechanicaldemands on these two tissues. Collagen II, a specific component ofcartilage ECM, is arranged in an open, intercrossing pattern which trapsglycosaminoglycans and facilitates resistance to compression.

[0005] The synthesis, secretion and assembly of collagens into specificsupramolecular aggregates is a complex, multi-step process. Fibrillarcollagens I, II and III are secreted as soluble procollagens comprisinga long, continuous triple helical “collagenous” region with smallerpolypeptide extensions (propeptides) at their amino (N) and carboxyl (C)ends. The removal of the propeptides by specific enzymes, the N- andC-propeptidases (proteinases), is a prerequisite for the correctassembly of collagens I and II into fibrils and for fibril growth. Theprocollagen C-propeptidase is identical to bone morphogenetic protein-1(BMP-1) and processes all three of these fibrillar collagens.Biochemically distinct N-propeptidases with specificity for procollagenI and procollagen II or for procollagen III are known. One bovine andhuman procollagen I/II N-propeptidases has been cloned. It is known asADAMTS-2.

[0006] The ADAMTS-2 protein (EC 3.4.24.14), also known as procollagenI/II amino-propetide processing enzyme or PCINP, catalyzes cleavage ofnative triple-helical procollagen I and procollagen II. The ADAMTS-2protein also has an affinity for collagen XIV. Lack of the ADAMTS-2protein is known to cause dermatosparaxis in cattle, or Ehlers-Danlossyndrome type VIIC (EDS-VIIC) in humans. EDS-VIIC is characterizedclinically by severe skin fragility, and biochemically by the presencein skin of procollagen which is incompletely processed at the aminoterminus. The molecular hallmark is the presence of irregular, thin,branched collagen fibrils in the dermis which appear “hieroglyphic” incross section and contain procollagen I with an intact N-propeptide,termed pN-collagen I. Similar findings have been described very recentlyin Adamts2 transgenic knockout mice. Thus, it is believed that theADAMTS-2 protein plays a role in processing of procollagen to maturecollagen, an essential step for correct assembly of collagen intocollagen fibrils.

[0007] A number of diseases have been shown to be caused by: synthesisof insufficient amounts of collagen; synthesis of defective collagen; orover-production of normal collagen in a form referred to as eitherfibrotic tissue or scars.

SUMMARY OF THE INVENTION

[0008] As used herein, the following abbreviations have the followingmeanings:

[0009] “a.a.” is used herein to mean amino acids;

[0010] “EDS” is used herein to mean Ehlers-Danlos syndrome;

[0011] “BMP-1” is used herin to mean bone morphogenetic protein-1;

[0012] “PCR” is used herein to mean polymerase chain reaction;

[0013] “RT-PCR” is used herein to mean reverse transcriptase-PCR;

[0014] “kbp” is used herein to mean kilobase pairs;

[0015] “bp” is used herein to mean base pairs;

[0016] “nt” is used herein to mean nucleotides;

[0017] “ECM” is used herein to mean extracellular matrix;

[0018] “TS” is used herein to mean thrombospondin;

[0019] “GAPDH” is used herein to mean Glyceraldehyde 3-phosphatedehydrogenase;

[0020] “UV” is used herein to mean ultraviolet.

[0021] Where appropriate, approved nomenclature is used for human andmouse genes. ADAMTS2 and ADAMTS3 are human genes. Adamts2 and Adamts3are the corresponding mouse orthologs. The protein products of therespective genes are designated ADAMTS-2 and ADAMTS-3. Trivial names forthe protein products are procollagen N-propeptidase 1 (PCNP1) andprocollagen N-propeptidase 2 (PCNP2).

[0022] The preferred nucleic acids of the invention are homologs andalleles of the nucleic acids of ADAMTS3. The invention further embracesfunctional equivalents, variants, analogs and fragments of the foregoingnucleic acids and also embraces proteins and peptides coded for by anyof the foregoing. For a discussion of what is meant by “variant” andother defined terms, reference is made to U.S. Pat. No. 6,391,610, whichis hereby incorporated in its entirety by reference thereto. The presentinvention also provides isolated polynucleotides which encode anADAMTS-3 protein or a variant thereof, polynucleotide sequencescomplementary to such polynucleotides, vectors containing suchpolynucleotides, and host cells transformed or transfected with suchvectors. The present invention also relates to antibodies which areimmunospecific for one or more of the ADAMTS-3 proteins.

[0023] According to one aspect of the invention, an isolated nucleicacid molecule is provided. The invention further embraces nucleic acidmolecules that differ from the foregoing isolated nucleic acid moleculesin codon sequence due to the degeneracy of the genetic code. Theinvention also embraces complements of the foregoing nucleic acids.

[0024] Preferred isolated nucleic acid molecules are those comprisingmammallian cDNAs or gene corresponding to ADAMTS3. Even more preferablythe present invention relates to isolated nucleic acid moleculescomprising human cDNA or genes corresponding to ADAMTS-3.

[0025] According to another aspect of the invention, isolatedpolypeptides (e.g. ADAMTS-3) coded for by the isolated nucleic acidmolecules described above also are provided as well as functionalequivalents, variants, analogs and fragments thereof. In one embodiment,the polypeptide is a human procollagen II N-propeptidase protein or afunctionally active fragment or variants thereof.

[0026] Another embodiment of the present invention relates to, isolated,substantially purified, mammalian proteins belonging to the ADAMTS-3subfamily. As used herein, the term “substantially purified” refers to aprotein that is removed from its natural environment, isolated orseparated, and at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which it is naturallyassociated.

[0027] Another embodiment of the present invention is comprised of amethod of preventing, treating or ameliorating a tissue-related disorderor condition in a patient comprising the steps of administering aneffective amount of ADAMTS3 or ADAMTS-3 to a site in need of repair,regeneration, or procollagen processing. This embodiment may preferablyinclude the steps of combining an effective amount of ADAMTS3 orADAMTS-3 and a delivery system to form a mixture; molding said mixtureto form an implant; and implanting said implant into said patient. Theimplant is preferably suitable as a tissue substitute, and even morepreferably is biodegradable. The delivery system may be comprised of anysuitable delivery system including natural polymers, systhetic polymers,collagen, hydroxyapatite, calcium phosphate ceramics, bioglass,hydrogels and mixtures thereof. The effective amount of ADAMTS-2 may bedelivered as a protein, a nucleic acid (e.g. a nucleic acid is selectedfrom the group consisting of a gene, a cDNA, a vector, an RNA molecule,an antisense molecule, a ribozyme, and a peptide nucleic acid (PNA)molecule).

[0028] Embodiments of the present invention also contemplate genetherapy, wherein defective cells of a donor are genetically engineeredto include an isolated nucleic acid expressing a functional ADAMTS-3protein. The cells are then returned to the donor.

[0029] One aspect of the present invention involves the discovery andisolation of the ADAMTS3 cDNA and the corresponding ADAMTS-3 protein Theexpression and biological activity of the proteins are believed to benecessary for normal procollagen processing, and alteration of theexpression or biological activity of these proteins may be used toinfluence propeptidase activity and thereby affect collagen properties.In addition, normal procollagen biosynthesis can be established bysupplying a nucleic acid expressing ADMATS-3.

[0030] The invention in another aspect involves a method for decreasingprocollagen propeptidase activity in a subject. An agent thatselectively binds to an isolated nucleic acid molecule described hereinor an expression product thereof is administered to a subject in need ofsuch treatment, in an amount effective to decrease procollagenpropeptidase activity in the subject. Preferred agents are modifiedantisense nucleic acids and polypeptides.

[0031] Nucleotide sequence submission. The partial sequence of ADAMTS3(the KIAA0366 gene) was previously reported by the Kazusa DNA Institutewith GenBank Accession Mo. AB002364. The novel sequence described herehas been submitted to GenBank and is available with Accession No.AF247668.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1A illustrates the domain organization of ADAMTS-3 andADAMTS-2 with a key for the domains shown at the bottom of the figure;

[0033]FIG. 1B illustrates alignment of the primary structures ofADAMTS-3 and ADAMTS-2 using the single-letter amino acid code;

[0034]FIG. 2A illustrates quantitative RT-PCR assay of MRNA levels ofADAMTS-3 and ADAMTS-2 in skin fibroblasts;

[0035]FIG. 2B illustrates quantitative RT-PCR assay of mRNA levels ofADAMTS-3 and ADAMTS-2 in human cartilage;

[0036]FIG. 3 illustrates that ADAMTS-3 excites the N-propeptide ofCollagen II;

[0037]FIG. 4 shows an amino acid sequence (SEQ ID NO: 1) of afull-length sequence for human ADAMTS-3;

[0038]FIG. 5 shows a nucleotide sequence (SEQ ID NO: 2) of a full-lengthhuman ADAMTS3 (coding region is in upper case) which encodes ADAMTS-3;

[0039]FIG. 6 shows an amino acid sequence (SEQ ID NO: 3) correspondingto the portion of human ADAMTS-3 corresponding to Accession No.AF247668;

[0040]FIG. 7 shows a Nucleotide sequence (SEQ ID NO. 4) corresponding tothe portion of ADAMTS3 which is necessary to encode ADAMTS-3.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0041] Among the various members of the ADAMTS family (currentlynumbering 20 gene products), the overall domain organization and aminoacid sequence of ADAMTS-2 is most analogous to ADAMTS-3. ADAMTS3 cDNAwas originally partially cloned from human brain and named the KIAA0366gene. The KIAA0366 cDNA was incomplete at the 5′ end, and thetranslation start codon had not been identified. Through molecularcloning described herein, the complete primary structure of ADAMTS3 isidentified herein as shown in SEQ ID NO: 2 and FIG. 5.

[0042] In light of the data presented here, showing that procollagen IIin dermatosparactic cartilage is completely processed, it apearsADAMTS-3 (complete sequence shown in SEQ ID NO: 1 and FIG. 4) isinvolved in procollagen II processing. The model system used was a Swarmrat chondrosarcoma derived cell line, RCS-LTC. In monolayer culture,these cells deposit an ECM containing collagens II, IX, and XI. However,the collagen is organized into thin filaments instead of fibrils.RCS-LTC cells fail to process procollagen II beyond the stage ofpN-collagen II, although the amino acid sequence of the N-propeptidasecleavage site in RCS-LTC procollagen II is normal. RCS-LTC pN-collagenII is, however, processed in vitro by addition of conditioned mediumfrom cultures of chick chondrocytes. This suggested that RCS-LTCchondrocytes either fail to express procollagen II N-propeptidase orlack a soluble, essential cofactor. RCS-LTC chondrocytes thus provide amodel system for identification of genes involved in procollagen IIamino propeptide processing.

[0043] A discussed in greater detail below, transfection of RCS-LTCcells with ADAMTS3 or ADAMTS2 results in conversion of a portion of thepN-collagen II to a fully processed form. The results establish thatN-propeptidase deficiency is responsible, at least in part, fordefective collagen processing in RCS-LTC cells. It also appears thatsteady-state mRNA levels of ADAMTS2 and ADAMTS3 are different in normalhuman skin, in skin fibroblasts, and in cartilage, with ADAMTS-3 beingexpressed at higher levels than ADAMTS-2 in cartilage. Together, thesedata suggest that ADAMTS-3 may be a major physiological procollagen IIN-propeptidase.

[0044] Thus, embodiments of the present invention may include ADAMTSsubfamily protein members, particularly those which are procollagenpropeptidases, genes encoding those proteins, functional modificationsand variants of the foregoing, useful fragments of the foregoing, aswell as therapeutics and diagnostics relating thereto. Moreparticularly, the present invention relates to ADAMTS-3 and ADAMTS3

[0045] Homologs and alleles of the procollagen N-propeptidase genes(e.g. ADAMTS3) of the invention can be identified by conventionaltechniques. Thus, an aspect of the invention is those nucleic acidsequences which code for procollagen N-propeptidase proteins and whichhybridize to a nucleic acid molecule consisting of ADAMTS 3, understringent conditions, preferably highly stringent conditions. The term“stringent conditions” as used herein refers to parameters with whichthe art is familiar, and reference is again made to U.S. Pat. No.6,391,610, ehich again, is incorporated herein in its entiret by refencethereto, particularly with regard to the definitions of “stringent” and“highliy stringent”. The skilled artisan will be familiar with suchconditions, and thus they are not given here. It will be understood,however, that the skilled artisan will be able to manipulate theconditions in a manner to permit the clear identification of homologsand alleles of procollagen N-propeptidase proteins of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

[0046] The invention also provides isolated unique fragments of ADAMTS3.A unique fragment is one that is a ‘signature’ for the larger nucleicacid. Unique fragments can be used as probes in Southern blot assays toidentify family members or can be used in amplification assays such asthose employing PCR. Unique fragments also can be used to produce fusionproteins for generating antibodies or for generating immunoassaycomponents. Likewise, unique fragments can be employed to producefragments of the procollagen N-propeptidase protein, useful, forexample, in immunoassays or as a competitive inhibitor of the substrateof the procollagen N-propeptidase protein in therapeutic applications.Unique fragments further can be used as antisense molecules to inhibitthe expression of the procollagen N-propeptidase proteins of theinvention, particularly for therapeutic purposes as described in greaterdetail below.

[0047] As mentioned above, the invention embraces antisenseoligonucleotides that selectively bind to a nucleic acid moleculeencoding an procollagen N-propeptidase protein, to decrease procollagenN-propeptidase activity (particularly procollagen I N-propeptidaseactivity). This is desirable in virtually any medical condition whereina reduction in collagen production activity is desirable.

[0048] As used herein, the term “antisense oligonucleotide” or“antisense” describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene.

[0049] Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

[0050] The invention also involves expression vectors coding forprocollagen N-propeptidase proteins (particularly procollagen II Npropeptidase proteins) and fragments and variants thereof and host cellscontaining those expression vectors. Virtually any cells, prokaryotic oreukaryotic, which can be transformed with heterologous DNA or RNA andwhich can be grown or maintained in culture, may be used in the practiceof the invention. Examples include bacterial cells such as E.coli andmammalian cells such as mouse, hamster, pig, goat, primate, etc. Theymay be of a wide variety of tissue types.

[0051] As used herein, a “vector” may be any of a number of nucleicacids into which a desired sequence may be inserted by restriction andligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids and phagemids. A cloning vector is one which isable to replicate in a host cell, and which is further characterized byone or more endonuclease restriction sites at which the vector may becut in a determinable fashion and into which a desired DNA sequence maybe ligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g. β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hots, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

[0052] As used herein, a coding sequence and regulatory sequences aresaid to be “operably” joined when they are covalently linked in such away as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalprotein, two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not: result in the introduction of aframe-shift mutation; interfere with the ability of the promoter regionto direct the transcription of the coding sequences; or interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a promoter region would be operably joined to a codingsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide.

[0053] Expression vectors containing all the necessary elements forexpression are commercially available and known to those skilled in theart. See Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding the procollagen N-propeptidase proteinor fragment or variant thereof. That heterologous DNA (RNA) is placedunder operable control of transcriptional elements to permit theexpression of the heterologous DNA in the host cell. In still anotheraspect of the invention, a defective chondrocyte or fibroblast orprecursor thereof is treated with DNA in a manner to promote viahomologous recombination intracellularly the correction of a defectiveprocollagen N-propeptidase gene.

[0054] The invention also contemplates screening assays to detect thepresence or absence of the procollagen N-propeptidase protein and inpurification protocols to isolate procollagen N-propeptidase proteins.

[0055] When used therapeutically, the compounds of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means that amount necessary to delaythe onset of, inhibit the progression of, or halt altogether theparticular condition being treated. Therapeutically effective amountsspecifically will be those which desirably influence procollagenN-propeptidase activity, be it inhibiting or enhancing procollagenN-propeptidase I or enhancing procollagen N propeptidase II. When it isdesired to decrease procollagen N-propeptidase I/II activity, then anyinhibition of procollagen N-propeptidase activity is regarded as atherapeutically effective amount. When it is desired to increaseprocollagen N-propeptidase I/II activity, then any enhancement ofprocollagen N-propeptidase activity is regarded as a therapeuticallyeffective amount. Generally, a therapeutically effective amount willvary with the subject's age, condition, and sex, as well as the natureand extent of the disease in the subject, all of which can be determinedby one of ordinary skill in the art. The dosage may be adjusted by theindividual physician or veterinarian, particularly in the event of anycomplication.

[0056] The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences;18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

[0057] The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is well known in the art. In general, itinvolves introduction in vitro of a functional copy of a gene into acell(s) of a subject which contains a defective copy of the gene, andreturning the genetically engineered cell(s) to the subject. Thefunctional copy of the gene is under operable control of regulatoryelements which permit expression of the gene in the geneticallyengineered cell(s). Numerous transfection and transduction techniques aswell as appropriate expression vectors are well known to those ofordinary skill in the art In vivo gene therapy using vectors such asadenovirus also is contemplated according to the invention.

[0058] In vivo gene therapy can also be used for systemic treatement, anarea in which gene therapy has broad applications. Systemic treatmentinvolves transfecting target cells with the DNA of interest, expressingthe coded protein in that cell, and the capability of the transformedcell to subsequently secrete the manufactured protein into blood.

[0059] A variety of methods have been developed to accomplish in vivotransformation including mechanical means (e.g. direct injection ofnucleic acid into target cells or particle bombardment), recombinantviruses, lipsomes, and receptor-mediated endocytosis (RME) (for reviews,see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr.14:328-37).

[0060] The following examples, materials, methods, experimentalprocedures, discussion, and detailed description are meant to furtherillustrates the present invention but, of course, should not beconstrued as in any way limiting its scope.

[0061] Materials and Methods, & Experimental Procedures: Cloning ofADAMTS3. The previously reported 5774 bp KIAA0366/ADAMTS3 cDNA (23) wasincomplete at its 5′. The sequence was extended further in the 5′direction by RACE using the Marathon™ system, and Marathon™ human fetalbrain cDNA (reagents from Clontech, Palo Alto) as template, essentiallyas previously described. PCR was done with nested ADAMTS3 specificantisense oligonucleotide primers 5′TCAAGGCCTTCCAGGTCCGACTCTC3′ and5′GGGAGCCTGTTCTACAGCTGATCTC3′ and with nested adaptor primers at the 5′end of the template. The RACE products were cloned and sequenced aspreviously described.

[0062] Generation of ADAMTS3 and ADAMTS2 expression constructs. Togenerate a cDNA construct for expression of full length ADAMTS-3, wefirst deleted the 5′ end of the KIAA0366 cDNA (in pBluescript II SK+[Stratagene, La Jolla], provided by Dr. Takahiro Nagase of the KazusaDNA Institute). The deleted segment extended from the 5′ Sal I cloning(i.e., vector) site up to a unique internal AccI site at nt position 598(KIAA0366 sequence enumeration). We replaced this fragment with aPCR-derived fragment of ADAMTS3 cDNA extending from the 5′ untranslatedsequence to just downstream of the AccI site. Briefly, PCR was performedwith Advantage PCR reagents (Clontech, Palo Alto, Calif.), using theRACE cDNA clone as template, the forward primer5′AACTCGAGGAAAGTGAACTCGACTCGTG3′ (XhoI site underlined) and reverseprimer 5′AGCCTGTTCTACAGCTGATC3′. The resulting amplicon was digestedwith XhoI and AccI (at the internal AccI site) and cloned into theSalI-AccI restricted KIAA0366 cDNA. This introduced the authenticADAMTS3 ribosome binding sequence, translation start codon and completesignal peptide into the KIAA0366 cDNA. This insert encoding full-lengthADAMTS-3 was excised from pBluescript with XhoI and NotI and ligatedinto the corresponding sites in pcDNA 3.1 (+) myc-His A (Invitrogen, SanDiego, Calif.). In this mammalian expression construct, ADAMTS-3 is notin frame with the C-terminal myc and poly-histidine tags. For expressionof ADAMTS-2, three overlapping bovine cDNA clones which have beenpreviously reported were appropriately digested (with NotI, BclI, EagI,and KpnI) and assembled to generate a construct encoding full-lengthbovine ADAMTS-2. The ADAMTS2 cDNA was inserted into the NotI/XbaI sitesof the pcDNA3 expression vector (Invitrogen, San Diego, Calif.) using aXbaI adapter. These were kindly provided by Dr. A. C. Colige.

[0063] Isolation of RNA. Skin samples obtained from the forearm ofhealthy volunteers were used to isolate dermal fibroblasts or werestored in liquid nitrogen until use (Laboratoire de Biologie des TissusConjonctifs, Sart-Tilman, Belgium, Ethics Committee ApprovalF94/14/1871). Dermal fibroblasts grown from skin explants werecultivated in Dulbecco's Minimum Essential Medium (DMEM) supplementedwith 10% fetal bovine serum (FBS). For northern analysis, total RNA waspurified from skin samples pulverized in liquid nitrogen or from dermalfibroblasts in culture, after solubilization of homogenates and cells in0.1M Tris HCl, pH 7.5, 5M guanidium thiocyanate, 1% β-mercaptoethanol.These extracts were centrifuged at 100,000×g for 18 h on a cesiumchloride (CsCl 2 ) cushion (0.01 M ethylene diamine tetra-acetic acid(EDTA), pH7.5, 5.7 M CsCl 2 ). MRNA was subsequently purified usingPolyATract mRNA Isolation System (Promega Benelux B.V.), according tomanufacturer's instructions. For quantitative RT-PCR, RNA was harvestedfrom human fetal cartilage or from dermal fibroblasts using Trizol (LifeTechnologies, Rockville, Md.) and manufacturer's recommended protocols.DNA was eliminated by treatment with DNase I (DNA-FreeTM, Ambion,Austin, Tex.).

[0064] Quantitative reverse transcriptase-PCR (RT-PCR) analysis. TotalRNA (2.5 mg) was reverse-transcribed with the SuperScript First-StrandSynthesis System for RT-PCR (Life Technologies, Rockville, Md.) usingoligo-dT as a primer. Real-time PCR was performed in an ABI Prism 7700Sequence Detector using SYBR Green PCR Core Reagents (AppliedBiosystems, Foster City, Calif.). In this system, continuous, automatedquantitation of the PCR product is performed by measuring thefluorescence generated by the binding of SYBR Green to double-strandedDNA. All PCR amplifications were performed in triplicate along withparallel measurements of GAPDH cDNA (an internal control). Data wereanalyzed according to the comparative C t method (Applied Biosystemsprotocols) and represented after normalization to GAPDH levels. Toconfirm these data, PCR products were also separated on a 2.0 % agarosegel, visualized with UV light through a SYBR Green filter andphotographed. The following primers were used for amplification at aconcentration of 300 nM: ADAMTS2,

[0065] 5′TGGGAAGCACAACGACATTG3′(forward) and

[0066] 5′CTCGGTCGTCGAGGGATTAG3′(reverse), ADAMTS3,

[0067] 5′TCAGTGGGAGGTCCAAATGCA3′(forward) and

[0068] 5′GCAAAGAAGGAAGCAGCAGCC3′(reverse), GAPDH,

[0069] 5′CCACTGCCAACGTGTCAGTGG3′(forward) and

[0070] 5′AAGGTGGAGGAGTGGGTGTCG3′(reverse). As an additional control,RT-PCR was also performed in the absence of template.

[0071] Northern analysis of ADAMTS2 and ADAMTS3 expression. Acommercially available adult human multiple tissue northern blot and amouse embryo northern blot (Clontech Inc. Palo Alto, Calif.) werehybridized as per manufacturer's instructions using ExpressHyb™hybridization fluid (Clontech, Palo Alto, Calif.). The following cDNAprobes were used after random-primed labeling with [α³²P]-dCTP: afragment containing nucleotides 946-1379 of human ADAMTS-2 cloned inpCR4-TOPO (for human multiple tissue northern blot); a 1.1 kbp HindIIIfragment from the KIAA0366/ADAMTS3 cDNA (for human multiple tissuenorthern blot); the insert of IMAGE clone 1246561, available withGenBank accession no. AA832579 (mouse ADAMTS-2 probe for mouse embryonorthern blot); the insert of IMAGE clone 727026, available withAccession no. AA402760 (mouse ADAMTS-3 probe for mouse embryo northernblot). Exposure of the blots to X-ray film was for 3-7 days.

[0072] Poly-A+ RNA (0.8 μg) from human skin or human skin fibroblastswas electrophoresed on a formaldehyde-agarose gel and blotted to HybondN⁺ nylon membrane (Amersham-Pharmacia Biotech). cRNA probes for humanADAMTS2 and ADAMTS3 were generated by transcription from the respectiveclones using the Strip-EZ RNA kit (Ambion), T3 RNA polymerase and[α³²P]-UTP as per manufacturer's instructions. Prehybridization (1 hour)and hybridization (18 hours) were performed at 65° C. in 0.2 M Na₂HPO₄(pH 7.2), 1 mM EDTA, 1% bovine serum albumin, 7% sodium dodecyl sulfate(SDS) and 20% formamide. Stringency washes were carried out at 65° C. in40 mM Na₂HPO₄, 1 mM EDTA and 1% SDS.

[0073] Cell Culture and stable transfection of RCS-LTC chondrocytes.Monolayer cultures of RCS-LTC cells were maintained in DMEM containing4.5 g/liter glucose (Life Technologies, Grand Island, N.Y.) and 10% FBS(Hyclone Labs, Logan, Utah) at 37° C. in 5% CO₂. Culture medium waschanged every other day and confluent cultures were sub-cultured every 2weeks as described previously. Lipofectamine Plus (Life Technologies,Grand Island, N.Y.) was used for RCS-LTC transfections. Cells wereplated at a density of 3×10⁵ cells/well in 6 well plates (FalconFranklin Lakes, N.J.). After 24 h, the wells were rinsed with OPTI-MEM(Life Technologies, Grand Island, N.Y.) and transfected with the humanADAMTS3 or bovine ADAMTS2 cDNA constructs mg DNA/well) as per themanufacturer's instructions. As a control for efficiency of transfectionand to provide a negative control for procollagen II processing, cellswere separately transfected with pcDNA3.1/Myc-His (+)lacZ encoding theE. coli lacZ gene. Mock transfections were performed without cDNA as acontrol for efficacy of antibiotic selection. After 4 days in culture,transfected cells were selected in media supplemented with 1 mg/ml G418sulfate (Geneticin, Life Technologies, Grand Island, N.Y.). After 2weeks in culture, the chondrocytes from the mock transfections did notsurvive selection. Geneticin-resistant ADAMTS3, ADAMTS2, and lacZtransfected chondrocytes were expanded and maintained as pools in serialmonolayer culture as previously described for the RCS-LTC cell line, butin the continued presence of 1 mg/ml Geneticin.

[0074] β-Galactosidase expression in the lacZ stable transfectants wasdetected histochemically by staining the cells with5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal, Life Technologies,Grand Island, N.Y.). Briefly, the cultures were rinsed withphosphate-buffered saline (150 mM NaCl, 15 mM sodium phosphate, pH 7.3),fixed with 0.2% glutaraldehyde and incubated with 1 mg/ml X-gal, 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM MgCl₂ inPBS for 24 h at 37° C.

[0075] Procollagen analysis in RCS-LTC cells. ADAMTS3, ADAMTS2 and lacZstable transfectants as well as untransfected RCS-LTC chondrocytes weregrown in 6 well plates (Falcon, Franklin Lakes, N.J.) followingsub-culture as described. For the last day of culture, the culturemedium was supplemented with 10 mg/ml L-ascorbate and 100 mg/mlβ-aminoproprionitrile. Cells and ECM were rinsed with PBS and extractedwith boiling Laemmli sample buffer containing 100 mM dithiothreitol(DTT) or with 0.4 M NaCl, 50 mM Tris HCl, pH 7.5 containing 5 mM EDTA, 1mM PMSF, 0.1 mM benzamidine, and 0.1% Triton X-100. To identifyprocollagen II processing intermediates, untransfected RCS-LTCchondrocytes were grown for 4 days in the absence of ascorbate andβ-aminopropionitrile. The cell layers were extracted as above. Extractswere repeatedly passed through a 27.5 G needle to reduce viscosity andwere centrifuged at 15,000 rpm for 30 minutes at 4° C.

[0076] Collagen II in extracts of cartilage and RCS-LTC transfectantswas analyzed by gel electrophoresis and immunoblotting. Extracts wereheated to 100° C. for 3 minutes in Laemmli sample buffer containing 100mM DTT. Collagen II chains were resolved by 6% polyacrylamide gelelectrophoresis and detected after transfer to polyvinyl difluoride(PVDF) membrane (BioRad, Hercules, Calif.) using a monoclonal antibodyto collagen II (1C10) at a dilution of 1:2000 and the Renaissancewestern blot detection reagent (NEN Lifescience Products, Boston,Mass.). Monoclonal antibody 1C10 (D. R. Eyre, unpublished data)recognizes an epitope in denatured α1(II) CB9, 7. It detects procollagenII as well as processed collagen II on western blots.

[0077] As controls, fully processed collagen II from a 4M guanidine HClextract of human fetal cartilage and pepsinized collagen II from theRCS-LTC cell line were used.

[0078] Procollagen cleavage analysis in dermatosparactic cartilage.Nasal cartilage from a dennatosparactic cow and normal fetal bovineepiphyseal cartilage were extracted in 4 M guanidine HCl, 50 mMTris-HCl, pH 7.4 with 2 mM EDTA, 5 mM benzamidine, 2 mM PMSF and 10 mM1,10 phenanthroline at 40° C. for 24 h. Collagen in this extract wasseparated by reducing SDS-PAGE gel electrophoresis and visualized byCoomassie blue staining and by western blot analysis using 1C10 asdescribed above.

[0079] Molecular cloning of full length ADAMTS-3 and comparison withADAMTS-2. Using 5′ RACE, a novel 720 bp cDNA clone encoding the 5′untranslated region,the translation start codon, signal peptide, andpro-domain of ADAMTS-3 was obtained. The novel 5′ sequence we obtainedhas not been previously described and is deposited in GenBank withAccession No. AF247668. When incorporated into the KIAA0366 sequence, wewere therefore able to identify the complete open reading frame (ORF)and conceptual translation product of ADAMTS3. The predicted start codonis the 5′ most methionine codon (ATG) in the ORF (FIG. 1B) and ispreceded by an in-frame stop codon, 16 nucleotides upstream. It islocated within the context of a suitable Kozak consensus sequence fortranslation (contains purine [G] at position −3 with respect to the A ofthe ATG codon, and G at position +4). The predicted ADAMTS-3 protein(1205 amino acids) is comparable in length to human and bovine ADAMTS-2(1211 and 1205 amino acids respectively). The predicted mature(furin-processed) forms of these proteases are also of comparablelength, 957 residues (ADAMTS-3) and 953 residues (bovine and humanADAMTS-2) long. ADAMTS3 predicts a full-length protein of molecular mass135.6 kDa and a furin processed form of 107.5 kDa. A number of N-linkedglycosylation sites are predicted by the sequence (see below and FIG. 1)and thus post-translational modification is likely to increase themolecular mass of ADAMTS-3.

[0080]FIG. 1A shows the amino acid identity (in percent) betweenADAMTS-3 and ADAMTS-2 for each domain (except the C-terminal domain). Ascan be seen in FIG. 1A, ADAMTS-3 and ADAMTS-2 have a similar domainstructure. As can also be seen in FIG. 1B, ADAMTS-3 and ADAMTS-2 haveand an overall sequence identity of 61%. From amino to carboxylterminus, each of these enzymes consists of the following domains (withpercent sequence identity in parentheses): signal peptide, pro-domain(37%) demarcated from the catalytic domain (85%) by a furin cleavagesite, disintegrin-like domain (77%), central thrombospondin type I (TS)repeat (63%), cysteine-rich domain (67%), spacer domain (56%), threeadditional TS repeats (64%), followed by a unique, mostly nonhomologousC-terminal extension. The likely furin processing site for generation ofmature ADAMTS-3 and ADAMTS-2 is indicated by the arrowhead in FIG. 1B.In FIG. 1B, the sequence encoded by the RACE clone is overlined; TSrepeats are underlined and indicated by numbers; the zinc-bindinghistidine triad is enclosed in a box; and the boundaries of a region ofcomplete amino acid sequence identity in the catalytic domain areindicated by the vertical arrows. The N-termini of the disintegrin-like(Dis) and spacer domains are indicated. The cysteine-rich domain extendsfrom TS repeat 1 to the start of the spacer domain. Potentialcell-binding RGD/E sequences are indicated by the thick overline. ThePLAC domains are shown by the dashed underline. Potential sites forN-linked glycosylation are indicated by asterisks.

[0081] The presumed furin cleavage sites in ADAMTS-3 and ADAMTS-2 are atidentical locations. ADAMTS-3 and ADAMTS-2 catalytic domains arestrikingly similar, demonstrating complete sequence identity (but forone amino acid) in a region of 69 amino acids which includes thezinc-binding active site sequence. ADAMTS-3 and ADAMTS-2 each have eightconsensus sites for potential N-linked glycosylation. Of these, four areconserved absolutely between ADAMTS-2 and ADAMTS-3, two conserved sitesbeing in the pro-domain, and one site each in the catalytic domain andthe second TS domain. In contrast to ADAMTS-2, which contains thepotential cell binding sequence CVRGDC, ADAMTS-3 has the sequence CVRGECat the corresponding location. The C-terminal domains of these twomolecules are of comparable length (184 and 191 amino acids in ADAMTS-2and ADAMTS-3, respectively), but show little sequence similarity otherthan a highly conserved PLAC (Protease and LACunin) domain (83%identity). The PLAC domain was first described in an insect protein,lacunin, which contains all the ancillary domains of ADAMTS, and cantherefore be considered an ADAMTS-like protein. The PLAC domains ofhuman ADAMTS-3 and bovine ADAMTS-2 each contain six cysteine residues.In a previously published human ADAMTS-2 sequence (GenBank Accession No.AJ003125), one of these cysteines (at position 1090 in FIG. 1B) wassubstituted by serine. However, it is likely that this represents asequence variation or error since there is a cysteine at this positionin three independent ADAMTS2 EST sequences (GenBank accession nos.AI417257, AI624388 and AI089232).

[0082] ADAMTS-2 and ADAMTS-3 constitute a structurally distinctiveprocollagen N-propeptidase (PCNP) subfamily of the ADAMTS family.ADAMTS-2 and ADAMTS-3, as a sub-group, are distinct from the rest of theADAMTS family in a number of ways. In terms of domain organization,these are the only two members of the ADAMTS family to have threeC-terminal TS domains. Furthermore, they are the only two members of theADAMTS family to have a substantial C-terminal extension downstream ofthese TS domains. The location of the PLAC domain within this C-terminalextension is also unique, because in other ADAMTS family members whereit is present, such as in ADAMTS-7B and ADAMTS-10 it is usually at thevery carboxyl end of the protein. In the PCNP sub-family, the PLACdomain is internal.

[0083] A number of sequence hallmarks are unique to the PCNP subfamilyof enzymes, including: the pro-domain contains only two cysteines, incontrast to other ADAMTS enzymes, which usually contain three; and thecatalytic domain contains 6 cysteines as opposed to eight for the otherADAMTS. In other members of the ADAMTS family, the usual arrangementconsists of five cysteines upstream of the zinc-binding site and threedownstream. In the PCNP subfamily, there are only three cysteinesupstream of the zinc-binding sequence. The three downstream cysteinesare however, at absolutely conserved positions with regard to otherADAMTS enymes. This arrangement of cysteines suggests that the catalyticdomain of the PCNP subfamily may be structurally different from that ofthe other ADAMTS enzymes. Additionally, the sequence of the zinc-bindingtriad (HETGHLGMEHD) in the PCNP subfamily is unique in that it containsthreonine in the third position (underlined), whereas all other ADAMTSenzymes have a hydrophobic residue with a long side-chain (leucine orisoleucine) at this position; and the spacer domains of the ADAMTSfamily are quite variable in length and sequence. However, those of thePCNP subfamily are significantly similar to each other (56% amino-acididentity).

[0084] On the basis of domain and amino acid sequence homology, thissubfamily appears to contain no more than three members as determined bya search of the completed human genome sequence (Celera Genomics,Rockville, Md.). We designate the “classical” PCNP, ADAMTS-2, as PCNP1,and ADAMTS-3 as PCNP2, in keeping with the practice of providing trivialnomenclature which reflects the substrate of the enzyme.

[0085] Differential tissue-specific expression of ADAMTS2 and ADAMTS3.Adamts2 and Adamts3 were both expressed during mouse embryogenesis.Adamts2 expression was noted in mouse embryos at 7, 15 and 17 days. TwomRNA species (7.8 kb and 4.0 kb) were detected. A single Adamts3 mRNAspecies (˜7.2 kb in size) was also detected in mouse embryos at 7, 15and 17 days of gestation. Restricted expression of ADAMTS2 and ADAMTS3was seen in the eight normal human adult tissues examined by northernanalysis. In human placenta, lung and liver, two ADAMTS2 transcriptswere present, migrating at approximately 7.8 kb and 4.0 kb as previouslydescribed. The previously described 2 kb transcript which encodes atruncated form of ADAMTS-2 was not detected. Highest expression ofADAMTS3 was noted in placenta with lower level expression in lung, brainand heart, with a single mRNA species migrating at approximately 7.0 kb.

[0086] In skin samples and in skin fibroblasts, northern analysis withequivalent amounts of cRNA probes for ADAMTS2 or ADAMTS3 demonstrated adifferential prevalence of steady-state mRNA levels. Autoradiogramsgenerated by 1 h (ADAMTS2) and 18 h exposure (ADAMTS-3) illustrated thispoint. Based on these different durations of exposure, it is concludedthat a substantially stronger signal is obtained with an ADAMTS2 probethan with ADAMTS3 in skin and skin fibroblasts, suggesting that theycontain higher steady-state levels of ADAMTS2 mRNA than ADAMTS3 mRNA.All three previously reported ADAMTS2 transcripts (7.0 kb, 4.0 kb and2.0 kb) as well as some other minor transcripts were seen in skinfibroblasts, while only the 4.0 kb and 2.0 kb mRNAs were found in skin.However, with the ADAMTS3 probe, a 4.8 kb band was identified, plus 2.3kb band in skin fibroblasts. The discrepancy of these ADAMTS-3 bandswith those noted in multiple tissue northerns is presently unexplained,but it is possible, that like ADAMTS2, ADAMTS3 also generates multipletranscripts in a tissue specific fashion. It was also noted that whileADAMTS3 signal is stronger in skin as opposed to skin fibroblasts, thereverse was true for ADAMTS2.

[0087] To obtain a quantitative estimate of relative mRNA levels,quantitative RT-PCR analysis of ADAMTS2 and ADAMTS3 mRNA in culturedskin fibroblasts and human cartilage was performed. The data demonstrateconsiderably higher levels of steady-state ADAMTS2 mRNA levels in humanskin fibroblasts relative to ADAMTS3. In contrast, as shown in FIG. 2Aand FIG. 2B quantitative RT-PCR analysis of RNA from human fetalcartilage demonstrates an approximately five-fold higher steady-statelevel of ADAMTS-3 mRNA compared to ADAMTS2. In FIGS. 2A and 2B, the meanand standard deviation of three different PCR reactions is shown, aswell as gel electrophoresis of representative PCR reactions (inset).

[0088] Procollagen II is completely processed in dermatosparacticcartilage. Dermatosparactic animals have no functioning ADAMTS-2.Despite this, Coomassie-blue staining of collagen extracted fromdermatosparactic nasal cartilage demonstrated the presence of collagenchains which migrated similarly to the processed collagen II chains incontrol cartilage. This was confirmed by western blot analysis of theseextracts using a monoclonal antibody which recognizes procollagen II aswell as processed collagen II. This analysis showed that essentially allof the immunoreactive collagen was fully processed. Some pN-collagen Iwas visible in the extracts from dermatosparactic cartilage, confirmingthe origin of this tissue.

[0089] ADAMTS3 and ADAMTS2 process procollagen II in transfected RCS-LTCcells. The efficiency of RCS-LTC transfection was monitored byβ-galactosidase staining. LacZ-transfected cells also served as anegative control for analysis of procollagen processing. FIG. 5A (panelsa and b) shows lacZ-transfected chondrocytes on day 2 and day 8 aftersubculture, following transfection, Geneticin selection andhistochemical staining for β-galactosidase activity. Cells stablytransfected with lacZ showed dark blue staining (representing about 10%of the population) while no staining was seen in the ADAMTS3 and ADAMTS2stably transfected populations as expected (FIG. 5A, panels c and d). Asimilar efficiency of transfection and/or expression was assumed for theADAMTS3- and ADAMTS2-transfected cells as for the lacZ-transfectedcells.

[0090] To determine if ADAMTS-3 was capable of enzymatically removingthe N-propeptide of procollagen II, lysates of ADAMTS3-, ADAMTS2- andlacZ-transfected RCS-LTC chondrocytes were blotted and procollagen IIand collagen II were identified using monoclonal antibody 1C10. Theresults show some processing of pN-collagen II to mature collagen II inthe ADAMTS2- and ADAMTS3-transfected cells, but none in lacZ-transfectedcells or in untransfected cells. Following N-propeptide excision, thea1(II) chains migrate faster than the pN-collagen II chains and at aposition similar to the naturally processed a1(III) chain or pepsinizedcollagen II.

[0091] Procollagen I processing in dermatosparaxis is most deficient inskin, although mature skin has some processed collagen. Many collagenI-containing dermatosparactic tissues such as tendon, ligament, scleraand aorta show the presence of significant amounts of fully processedcollagen I. None of these tissues, nor bone, which relies on collagen Ifor its mechanical strength, have been noted to be fragile. Veryrecently, Adamts2 knockout mice have been reported to have significantamounts of processed collagen in skin.

[0092] These anomalies were attributed to the presence of residualpN-collagen processing activity, due to either the incompleteness of thegenetic defect, or to compensation by another enzyme. The demonstrationthat the causative mutations were functionally null favored theexistence of one or more additional procollagen N-propeptidases. Thepresence of processed procollagen I in many dermatosparactic tissues,including skin, suggested that this putative alternative propeptidase(s)might be regulated differently from ADAMTS-2 in skin and other tissuesor that it may not be as efficient in procollagen I processing asADAMTS-2.

[0093] ADAMTS-2 can process procollagen II. Therefore, we expected thatprocollagen II processing would be abnormal in dermatosparacticcartilage. However, it appears that collagen II is normally processed indermatosparactic cartilage, despite the absence of ADAMTS-2. This leadsto the conclusion that enzyme(s) other than ADAMTS-2 that could processprocollagen II exist (implication ADAMTS-3). Although EDS-VIIC patientsare of short stature, they do not have chondrodysplasia or prematurearthritis. A defect in collagen II processing similar to the collagen Iprocessing defect in dermatosparactic skin would be expected to cause asevere chondrodysplasia, given the critical role of collagen II instructural stability of cartilage matrix. Our studies provide anexplanation for the absence of cartilage fragility and/orchondrodysplasia in dermatosparaxis or EDS-VIIC. Our data suggests thepresence of an alternative pathway of procollagen II processing. Thepresence of procollagen I in dermatosparactic nasal cartilage, but notin normal articular cartilage may be explained by the differences incomposition of these cartilages, the inclusion of perichondrium in theextract, or by upregulation of collagen I gene expression, which hasbeen previously noted in dermatosparaxis. In contrast to our finding,the recently described Adamts2 knockout mice retain some unprocessedcollagen II in their cartilage.

[0094] ADAMTS-2 and ADAMTS-3 comprise a structurally and functionallydistinct subfamily of ADAMTS proteases. ADAMTS-3 and ADAMTS-2 areclosely related in the length of the polypeptide chains, and theirprimary sequence and domain organization, but are located on differentchromosomes. We previously mapped ADAMTS3 to human chromosome 4,distinct from the ADAMTS2 locus on human chromosome 5. While the closesimilarities in their catalytic domains suggest similar catalyticmechanisms, greater differences in their ancillary domains (i.e., theTS, disintegrin-like, cysteine rich and spacer domains) may affectsubstrate preferences or binding and compartmentalization in ECM. Forexample, ADAMTS-1 ancillary domains are responsible for ECM-binding andthe TS domains of aggrecanase-1 (ADAMTS-4) are required for binding tonative aggrecan. A splice variant of ADAMTS2 which generates a shortform of ADAMTS-2 lacking the ancillary domains is functionally inactivein procollagen I processing.

[0095] Gene regulation can also determine which of two or more relatedgenes are functional in any given tissue. Multiple tissue northern blotsdemonstrated that ADAMTS2 and ADAMTS3 are differentially regulated invarious tissues. Our results indicated that the steady-state mRNA levelsof ADAMTS2 were substantially higher than ADAMTS-3 in RNA isolated fromskin and even more so in RNA isolated from skin fibroblasts. Data fromnorthern blot analysis was supported by real-time, quantitative RT-PCRanalysis of skin fibroblast RNA. In contrast, in human cartilage,ADAMTS-3 levels were over four-fold higher than those of ADAMTS-2.

[0096] Transfection with ADAMTS2 and ADAMTS3 leads to procollagen IIprocessing in RCS-LTC cells. We have taken a genetic approach toidentify a function for ADAMTS3 and to determine the nature of theunderlying processing defect in RCS-LTC cells. The system was RCS-LTCcell line with stable transfection (pools) and collagen II Mab 1C10(Eyre). As shown in FIG. 3, transfection of these cells with ADAMTS2 andADAMTS3, but not with lacZ (a negative control), results in processingof pN-collagen II to the fully processed form. The controls were (+)+Pepsinized RCS-LTC collagen; +Fetal collagen II; −RCS-LTC collagen II;and +ADAMTS-2 transfection. Generation of processed collagen II wasevidenced by co-migration of the processed form with pepsinized RCS-LTCprocollagen II (pepsin removes noncollagenous propeptides) and withnative collagen II isolated from human cartilage. This suggests thatADAMTS-3, like ADAMTS-2, has pN-collagen II processing activity. Ourresults suggest that in quantitative terms it is roughly equivalent tothat of ADAMTS-2. Given the fact that ADAMTS-2 is an establishedprocollagen I/II processing enzyme and that there is a high degree ofsimilarity of ADAMTS-3 to ADAMTS-2, it is very likely that ADAMTS-3directly processes procollagen II. It may also serve as a target forinhibitors or competitors of ADAMTS-2.

[0097] There are several possible explanations for the persistence ofsubstantial amounts of pN-collagen II in ADAMTS2- or ADAMTS3-transfectedcells. The transfected cells were maintained as a pooled populationrather than as clonally selected lines so that there may be transfectedcells which do not express the construct at all or do so at low levels.To support this possibility, only a small proportion of lacZ-transfectedcells was found to express β-galactosidase activity. While not wishingto be bound by theory, it is unlikely that cells not containing the cDNAconstructs survive because control, untransfected cells subjected toGeneticin selection pressure were killed after 2 weeks.

[0098] The defect in RCS-LTC cells suggests a failure to produce afunctional processing enzyme. This could result from a structuralmutation in ADAMTS-2 or ADAMTS-3 or because of transcriptionalrepression of these genes in RCS-LTC cells. While ot wishing to be boundby theory, it is proposed that, either on the basis of substratepreference for procollagen II in vivo or on the basis of higherexpression in cartilage than ADAMTS-2, ADAMTS-3 is the principalcollagen II N-propeptidase in vivo. This is supported by data orobservations, including:there is processing of procollagen II despite anull mutation in ADAMTS2; there is roughly equivalent processing ofcollagen II in transfected RCS-LTC cells by ADAMTS-2 or ADAMTS-3; thereare greater than four-fold higher levels of ADAMTS3 than ADAMTS2 MRNA inhuman cartilage.

[0099] There may be other enzymes that contribute substantially tocollagen processing in tissues other than skin. To this end, theexistence a new member of the PCNP subfamily (ADAMTS-14) was recentlyidentified. Its role in procollagen processing has not yet been studied.

[0100] It is proposed herein that ADAMTS-2 and ADAMTS-3 both processprocollagen II, but ADAMTS-3 is likely to be more physiologicallyrelevant in this context, possibly due to cartilage-preferredexpression. Our data provide insight into the sparing of cartilage and,perhaps, into the relative sparing of some procollagen I-containingtissues in dermatosparaxis. Since it has been shown that ADAMTS-3 willprocess procollagen II, it is also likely that it will processprocollagen I. This is supported by the fact that the related genefamily member ADAMTS2 can process both procollagen I and procollagen II.The data presented herein illustrate that ADAMTS-2 and ADAMTS-3 areroughly equivalent in terms of procollagen II processing. Finally, theamino acid cleavage sequence in procollagen I and procollagen II areidentical.

[0101] Fibrosis and scarring are conditions in which excess fibroustissue (comprising mainly collagen) is produced following injury,inflammation or attempted repair as for example after surgical incision.In such conditions, the stability and structure of the excess collagencan be rendered inferior and thus less disruptive by preventing theaction of ADAMTS-3. Since removal of the N-propeptide is a requirementfor formation of collagen fibers and development of an organizedcollagenous matrix, inhibition of ADAMTS-3 by any means constitutes apotential interference with the fibrosis process and makes ADAMTS-3 adisease or drug target. There are also applications where it may benecessary to introduce or elevate levels of ADAMTS-3. In reconstructingtissues in the art known as tissue engineering, as for example ofcartilage, it will be essential to have completely processed procollagenII for optimal organization and function of collagen II. In this case,ADAMTS-3 may be introduced into the tissue engineered product by avariety of means to provide tissue engineered cartilage of optimalfunction.

[0102] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations of the preferred compounds and methods may beused and that it is intended that the invention may be practicedotherwise than as specifically described herein.

1 4 1 1205 PRT Homo sapiens 1 Met Val Leu Leu Ser Leu Trp Leu Ile AlaAla Ala Leu Val Glu Val 1 5 10 15 Arg Thr Ser Ala Asp Gly Gln Ala GlyAsn Glu Glu Met Val Gln Ile 20 25 30 Asp Leu Pro Ile Lys Arg Tyr Arg GluTyr Glu Leu Val Thr Pro Val 35 40 45 Ser Thr Asn Leu Glu Gly Arg Tyr LeuSer His Thr Leu Ser Ala Ser 50 55 60 His Lys Lys Arg Ser Ala Arg Asp ValSer Ser Asn Pro Glu Gln Leu 65 70 75 80 Phe Phe Asn Ile Thr Ala Phe GlyLys Asp Phe His Leu Arg Leu Lys 85 90 95 Pro Asn Thr Gln Leu Val Ala ProGly Ala Val Val Glu Trp His Glu 100 105 110 Thr Ser Leu Val Pro Gly AsnIle Thr Asp Pro Ile Asn Asn His Gln 115 120 125 Pro Gly Ser Ala Thr TyrArg Ile Arg Lys Thr Glu Pro Leu Gln Thr 130 135 140 Asn Cys Ala Tyr ValGly Asp Ile Val Asp Ile Pro Gly Thr Ser Val 145 150 155 160 Ala Ile SerAsn Cys Asp Gly Leu Ala Gly Met Ile Lys Ser Asp Asn 165 170 175 Glu GluTyr Phe Ile Glu Pro Leu Glu Arg Gly Lys Gln Met Glu Glu 180 185 190 GluLys Gly Arg Ile His Val Val Tyr Lys Arg Ser Ala Val Glu Gln 195 200 205Ala Pro Ile Asp Met Ser Lys Asp Phe His Tyr Arg Glu Ser Asp Leu 210 215220 Glu Gly Leu Asp Asp Leu Gly Thr Val Tyr Gly Asn Ile His Gln Gln 225230 235 240 Leu Asn Glu Thr Met Arg Arg Arg Arg His Ala Gly Glu Asn AspTyr 245 250 255 Asn Ile Glu Val Leu Leu Gly Val Asp Asp Ser Val Val ArgPhe His 260 265 270 Gly Lys Glu His Val Gln Asn Tyr Leu Leu Thr Leu MetAsn Ile Val 275 280 285 Asn Glu Ile Tyr His Asp Glu Ser Leu Gly Val HisIle Asn Val Val 290 295 300 Leu Val Arg Met Ile Met Leu Gly Tyr Ala LysSer Ile Ser Leu Ile 305 310 315 320 Glu Arg Gly Asn Pro Ser Arg Ser LeuGlu Asn Val Cys Arg Trp Ala 325 330 335 Ser Gln Gln Gln Arg Ser Asp LeuAsn His Ser Glu His His Asp His 340 345 350 Ala Ile Phe Leu Thr Arg GlnAsp Phe Gly Pro Ala Gly Met Gln Gly 355 360 365 Tyr Ala Pro Val Thr GlyMet Cys His Pro Val Arg Ser Cys Thr Leu 370 375 380 Asn His Glu Asp GlyPhe Ser Ser Ala Phe Val Val Ala His Glu Thr 385 390 395 400 Gly His ValLeu Gly Met Glu His Asp Gly Gln Gly Asn Arg Cys Gly 405 410 415 Asp GluThr Ala Met Gly Ser Val Met Ala Pro Leu Val Gln Ala Ala 420 425 430 PheHis Arg Tyr His Trp Ser Arg Cys Ser Gly Gln Glu Leu Lys Arg 435 440 445Tyr Ile His Ser Tyr Asp Cys Leu Leu Asp Asp Pro Phe Asp His Asp 450 455460 Trp Pro Lys Leu Pro Glu Leu Pro Gly Ile Asn Tyr Ser Met Asp Glu 465470 475 480 Gln Cys Arg Phe Asp Phe Gly Val Gly Tyr Lys Met Cys Thr AlaPhe 485 490 495 Arg Thr Phe Asp Pro Cys Lys Gln Leu Trp Cys Ser His ProAsp Asn 500 505 510 Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro Leu AspGly Thr Glu 515 520 525 Cys Ala Ala Gly Lys Trp Cys Tyr Lys Gly His CysMet Trp Lys Asn 530 535 540 Ala Asn Gln Gln Lys Gln Asp Gly Asn Trp GlySer Trp Thr Lys Phe 545 550 555 560 Gly Ser Cys Ser Arg Thr Cys Gly ThrGly Val Arg Phe Arg Thr Arg 565 570 575 Gln Cys Asn Asn Pro Met Pro IleAsn Gly Gly Gln Asp Cys Pro Gly 580 585 590 Val Asn Phe Glu Tyr Gln LeuCys Asn Thr Glu Glu Cys Gln Lys His 595 600 605 Phe Glu Asp Phe Arg AlaGln Gln Cys Gln Gln Arg Asn Ser His Phe 610 615 620 Glu Tyr Gln Asn ThrLys His His Trp Leu Pro Tyr Glu His Pro Asp 625 630 635 640 Pro Lys LysArg Cys His Leu Tyr Cys Gln Ser Lys Glu Thr Gly Asp 645 650 655 Val AlaTyr Met Lys Gln Leu Val His Asp Gly Thr His Cys Ser Tyr 660 665 670 LysAsp Pro Tyr Ser Ile Cys Val Arg Gly Glu Cys Val Lys Val Gly 675 680 685Cys Asp Lys Glu Ile Gly Ser Asn Lys Val Glu Asp Lys Cys Gly Val 690 695700 Cys Gly Gly Asp Asn Ser His Cys Arg Thr Val Lys Gly Thr Phe Thr 705710 715 720 Arg Thr Pro Arg Lys Leu Gly Tyr Leu Lys Met Phe Asp Ile ProPro 725 730 735 Gly Ala Arg His Val Leu Ile Gln Glu Asp Glu Ala Ser ProHis Ile 740 745 750 Leu Ala Ile Lys Asn Gln Ala Thr Gly His Tyr Ile LeuAsn Gly Lys 755 760 765 Gly Glu Glu Ala Lys Ser Arg Thr Phe Ile Asp LeuGly Val Glu Trp 770 775 780 Asp Tyr Asn Ile Glu Asp Asp Ile Glu Ser LeuHis Thr Asp Gly Pro 785 790 795 800 Leu His Asp Pro Val Ile Val Leu IleIle Pro Gln Glu Asn Asp Thr 805 810 815 Arg Ser Ser Leu Thr Tyr Lys TyrIle Ile His Glu Asp Ser Val Pro 820 825 830 Thr Ile Asn Ser Asn Asn ValIle Gln Glu Glu Leu Asp Thr Phe Glu 835 840 845 Trp Ala Leu Lys Ser TrpSer Gln Val Ser Lys Pro Cys Gly Gly Gly 850 855 860 Phe Gln Tyr Thr LysTyr Gly Cys Arg Arg Lys Ser Asp Asn Lys Met 865 870 875 880 Val His ArgSer Phe Cys Glu Ala Asn Lys Lys Pro Lys Pro Ile Arg 885 890 895 Arg MetCys Asn Ile Gln Glu Cys Thr His Pro Leu Trp Val Ala Glu 900 905 910 GluTrp Glu His Cys Thr Lys Thr Cys Gly Ser Ser Gly Tyr Gln Leu 915 920 925Arg Thr Val Arg Cys Leu Gln Pro Leu Leu Asp Gly Thr Asn Arg Ser 930 935940 Val His Ser Lys Tyr Cys Met Gly Asp Arg Pro Glu Ser Arg Arg Pro 945950 955 960 Cys Asn Arg Val Pro Cys Pro Ala Gln Trp Lys Thr Gly Pro TrpSer 965 970 975 Glu Cys Ser Val Thr Cys Gly Glu Gly Thr Glu Val Arg GlnVal Leu 980 985 990 Cys Arg Ala Gly Asp His Cys Asp Gly Glu Lys Pro GluSer Val Arg 995 1000 1005 Ala Cys Gln Leu Pro Pro Cys Asn Asp Glu ProCys Leu Gly Asp 1010 1015 1020 Lys Ser Ile Phe Cys Gln Met Glu Val LeuAla Arg Tyr Cys Ser 1025 1030 1035 Ile Pro Gly Tyr Asn Lys Leu Cys CysGlu Ser Cys Ser Lys Arg 1040 1045 1050 Ser Ser Thr Leu Pro Pro Pro TyrLeu Leu Glu Ala Ala Glu Thr 1055 1060 1065 His Asp Asp Val Ile Ser AsnPro Ser Asp Leu Pro Arg Ser Leu 1070 1075 1080 Val Met Pro Thr Ser LeuVal Pro Tyr His Ser Glu Thr Pro Ala 1085 1090 1095 Lys Lys Met Ser LeuSer Ser Ile Ser Ser Val Gly Gly Pro Asn 1100 1105 1110 Ala Tyr Ala AlaPhe Arg Pro Asn Ser Lys Pro Asp Gly Ala Asn 1115 1120 1125 Leu Arg GlnArg Ser Ala Gln Gln Ala Gly Ser Lys Thr Val Arg 1130 1135 1140 Leu ValThr Val Pro Ser Ser Pro Pro Thr Lys Arg Val His Leu 1145 1150 1155 SerSer Ala Ser Gln Met Ala Ala Ala Ser Phe Phe Ala Ala Ser 1160 1165 1170Asp Ser Ile Gly Ala Ser Ser Gln Ala Arg Thr Ser Lys Lys Asp 1175 11801185 Gly Lys Ile Ile Asp Asn Arg Arg Pro Thr Arg Ser Ser Thr Leu 11901195 1200 Glu Arg 1205 2 5822 DNA Homo sapiens 2 gctttgccca gtagttggaaagtgaactcg actcgtgatg gttctcctgt cactttggtt 60 gatagcagcc gctctggtagaggttaggac ttcagctgat ggacaagctg gtaatgaaga 120 aatggtgcaa atagatttaccaataaagag atatagagag tatgagctgg tgactccagt 180 cagcacaaat ctagaaggacgctatctctc ccatactctt tctgcgagtc acaaaaagag 240 gtcagcgagg gacgtgtcttccaaccctga gcagttgttc tttaacatca cggcatttgg 300 aaaagatttt catctgcgactaaagcccaa cactcaacta gtagctcctg gggctgttgt 360 ggagtggcat gagacatctctggtgcctgg gaatataacc gatcccatta acaaccatca 420 accaggaagt gctacgtatagaatccggaa aacagagcct ttgcagacta actgtgctta 480 tgttggtgac atcgtggacattccaggaac ctctgttgcc atcagcaact gtgatggtct 540 ggctggaatg ataaaaagtgataatgaaga gtatttcatt gaacccttgg aaagaggtaa 600 acagatggag gaagaaaaaggaaggattca tgttgtctac aagagatcag ctgtagaaca 660 ggctcccata gacatgtccaaagacttcca ctacagagag tcggacctgg aaggccttga 720 tgatctaggt actgtttatggcaacatcca ccagcagctg aatgaaacaa tgagacgccg 780 cagacacgcg ggagaaaacgattacaatat cgaggtactg ctgggagtgg atgactctgt 840 ggtccgtttc catggcaaagagcacgtcca aaactacctc ctgaccctaa tgaacattgt 900 gaatgaaatt taccatgatgagtccctcgg agtgcatata aatgtggtcc tggtgcgcat 960 gataatgctg ggatatgcaaagtccatcag cctcatagaa aggggaaacc catccagaag 1020 cttggagaat gtgtgtcgctgggcgtccca acagcaaaga tctgatctca accactctga 1080 acaccatgac catgcaatttttttaaccag gcaagacttt ggacctgctg gaatgcaagg 1140 atatgctcca gtcaccggcatgtgtcatcc agtgagaagt tgtaccctga atcatgagga 1200 tggtttttca tctgcttttgtagtagccca tgaaacgggc catgtgttgg gaatggagca 1260 tgatggacaa ggcaacaggtgtggtgatga gactgctatg ggaagtgtca tggctccctt 1320 ggtacaagca gcattccatcgttaccactg gtcccgatgc agtggtcaag aactgaaaag 1380 atatatccat tcctatgactgtctccttga tgaccctttt gatcatgatt ggcctaaact 1440 cccagaactt cctggaatcaattattctat ggatgagcaa tgtcgttttg attttggtgt 1500 tggctataaa atgtgcaccgcgttccgaac ctttgaccca tgtaaacagc tgtggtgtag 1560 ccatcctgat aatccctacttttgtaagac taaaaaggga cctccacttg atgggactga 1620 atgtgctgct ggaaaatggtgctataaggg tcattgcatg tggaagaatg ctaatcagca 1680 aaaacaagat ggcaattgggggtcatggac taaatttggc tcctgttctc ggacatgtgg 1740 aactggtgtt cgtttcagaacacgccagtg caataatccc atgcccatca atggtggtca 1800 ggattgtcct ggtgttaattttgagtacca gctttgtaac acagaagaat gccaaaaaca 1860 ctttgaggac ttcagagcacagcagtgtca gcagcgaaac tcccactttg aataccagaa 1920 taccaaacac cactggttgccatatgaaca tcctgacccc aagaaaagat gccaccttta 1980 ctgtcagtcc aaggagactggagatgttgc ttacatgaaa caactggtgc atgatggaac 2040 gcactgttct tacaaagatccatatagcat atgtgtgcga ggagagtgtg tgaaagtggg 2100 ctgtgataaa gaaattggttctaataaggt tgaggataag tgtggtgtct gtggaggaga 2160 taattcccac tgccgaaccgtgaaggggac atttaccaga actcccagga agcttgggta 2220 ccttaagatg tttgatataccccctggggc tagacatgtg ttaatccaag aagacgaggc 2280 ttctcctcat attcttgctattaagaacca ggctacaggc cattatattt taaatggcaa 2340 aggggaggaa gccaagtcgcggaccttcat agatcttggt gtggagtggg attataacat 2400 tgaagatgac attgaaagtcttcacaccga tggaccttta catgatcctg ttattgtttt 2460 gattatacct caagaaaatgatacccgctc tagcctgaca tataagtaca tcatccatga 2520 agactctgta cctacaatcaacagcaacaa tgtcatccag gaagaattag atacttttga 2580 gtgggctttg aagagctggtctcaggtttc caaaccctgt ggtggaggtt tccagtacac 2640 taaatatgga tgccgtaggaaaagtgataa taaaatggtc catcgcagct tctgtgaggc 2700 caacaaaaag ccgaaacctattagacgaat gtgcaatatt caagagtgta cacatccact 2760 ctgggtagca gaagaatgggaacactgcac caaaacctgt ggaagttctg gctatcagct 2820 tcgcactgta cgctgccttcagccactcct tgatggcacc aaccgctctg tgcacagcaa 2880 atactgcatg ggtgaccgtcccgagagccg ccggccctgt aacagagtgc cctgccctgc 2940 acagtggaaa acaggaccctggagtgagtg ttcagtgacc tgcggtgaag gaacggaggt 3000 gaggcaggtc ctctgcagggctggggacca ctgtgatggt gaaaagcctg agtcggtcag 3060 agcctgtcaa ctgcctccttgtaatgatga accatgtttg ggagacaagt ccatattctg 3120 tcaaatggaa gtgttggcacgatactgctc cataccaggt tataacaagt tatgttgtga 3180 gtcctgcagc aagcgcagtagcaccctgcc accaccatac cttctagaag ctgctgaaac 3240 tcatgatgat gtcatctctaaccctagtga cctccctaga tctctagtga tgcctacatc 3300 tttggttcct tatcattcagagacccctgc aaagaagatg tctttgagta gcatctcttc 3360 agtgggaggt ccaaatgcatatgctgcttt caggccaaac agtaaacctg atggtgctaa 3420 tttacgccag aggagtgctcagcaagcagg aagtaagact gtgagactgg tcaccgtacc 3480 atcctcccca cccaccaagagggtccacct cagttcagct tcacaaatgg ctgctgcttc 3540 cttctttgca gccagtgattcaataggtgc ttcttctcag gcaagaacct caaagaaaga 3600 tggaaagatc attgacaacagacgtccgac aagatcatcc accttagaaa gatgagaaag 3660 tgaaccaaaa aggctagaaaccagaggaaa acctggacaa cctctctctt cccatggtgc 3720 atatgcttgt ttaaagtggaaatctctata gatcgtcagc tcattttatc tgtaattgga 3780 agaacagaaa gtgctggctcactttctagt tgctttcatc ctccttttgt tctgcattga 3840 ctcatttacc agaattcattggaagaaatc accaaagatt attacaaaag aaaaatatgt 3900 tgctaagatt gtgttggtcgctctctgaag cagaaaaggg actggaacca attgtgcata 3960 tcagctgact ttttgtttgttttagaaaag ttacagtaaa aattaaaaag agataccaat 4020 ggtttacact ttaacaagaaattttggata tggaacaaag aattcttaga cttgtattcc 4080 tatttatcta tattagaaatattgtatgag caaatttgca gctgttgtgt aaatactgta 4140 tattgcaaaa atcagtattattttaagaga tgtgttctca aatgattgtt tactatatta 4200 catttctgga tgttctaggtgcctgtcgtt gagtattgcc ttgtttgaca ttctataggt 4260 taattttcaa agcagagtattacaaaagag aagttagaat tacagctact gacaatataa 4320 agggttttgt tgaatcaacaatgtgatacg taaattatag aaaaagaaaa gaaacacaaa 4380 agctatagat atacagatatcagcttacct attgccttct atacttataa tttaaaggat 4440 tggtgtctta gtacacttgtggtcacaggg atcaacgaat agtaaataat gaactcgtgc 4500 aagacaaaac tgaaaccctctttccaggac ctcagtaggc accgttgagg tgtcctttgt 4560 ttttgtgtgt gtgtgttcttttttaatttt cgcattgttg acagatacaa acagttatac 4620 tcaatgtact gtaataatcgcaaaggaaaa agttttggga taacttattt gtatgttggt 4680 agctgagaaa aatatcatcagtctagaatt gatatttgag tatagtagag ctttggggct 4740 ttgaaggcag gttcaagaaagcatatgtcg atggttgaga tatttatttt ccatatggtt 4800 catgttcaaa tgttcacaaccacaatgcat ctgactgcaa taatgtgcta ataatttatg 4860 tcagtagtca ccttgctcacagcaaagcca gaaatgctct ctccagggag tagatgtaaa 4920 gtacttgtac atagaattcagaactgaaga tatttattaa aagttgattt ttttttcttg 4980 atagtatttt tatgtactaaatatttacac taatatcaat tacatatttt ggtaaactag 5040 agagacataa ttagagatgcatgctttgtt ctgtgcatag agacctttaa gcaaactact 5100 acagccaact caaaagctaaaactgaacaa atttgatgtt atgcaaacat cttgcatttt 5160 tagtagttga tattaagttgatgacttgtt tcccttcaag gaaacattaa attgtatgga 5220 ctcagctagc tgttcaatgaaattgtgaat tagaaacatt tttaaaagtt tttgaaagag 5280 ataagtgcat catgaattacatgtacatga gaggagatag tgatatcagc ataatgattt 5340 tgaggtcagt acctgagctgtctaaaaata tattatacaa actaaaatgt agatgaatta 5400 acctctcaaa gcacagaatgtgcaagaact tttgcatttt aatcgttgta aactaacagc 5460 ttaaactatt gactctatacctctaaagaa ttgctgctac tttgtgcaag aactttgaag 5520 gtcaaattag gcaaattccagatagtaaaa caatccctaa gccttaagtc tttttttttt 5580 cctaaaaatt cccatagaataaaattctct ctagtttact tgtgtgtgca tacatctcat 5640 ccacagggga agataaagatggtcacacaa acagtttcca taaagatgta catattcatt 5700 atacttctga cctttgggctttcttttcta ctaagctaaa aattcctttt tatcaaagtg 5760 tacactactg atgctgtttgttgtactgag agcacgtacc aataaaaatg ttaacaaaat 5820 at 5822 3 227 PRT Homosapiens 3 Met Val Leu Leu Ser Leu Trp Leu Ile Ala Ala Ala Leu Val GluVal 1 5 10 15 Arg Thr Ser Ala Asp Gly Gln Ala Gly Asn Glu Glu Met ValGln Ile 20 25 30 Asp Leu Pro Ile Lys Arg Tyr Arg Glu Tyr Glu Leu Val ThrPro Val 35 40 45 Ser Thr Asn Leu Glu Gly Arg Tyr Leu Ser His Thr Leu SerAla Ser 50 55 60 His Lys Lys Arg Ser Ala Arg Asp Val Ser Ser Asn Pro GluGln Leu 65 70 75 80 Phe Phe Asn Ile Thr Ala Phe Gly Lys Asp Phe His LeuArg Leu Lys 85 90 95 Pro Asn Thr Gln Leu Val Ala Pro Gly Ala Val Val GluTrp His Glu 100 105 110 Thr Ser Leu Val Pro Gly Asn Ile Thr Asp Pro IleAsn Asn His Gln 115 120 125 Pro Gly Ser Ala Thr Tyr Arg Ile Arg Lys ThrGlu Pro Leu Gln Thr 130 135 140 Asn Cys Ala Tyr Val Gly Asp Ile Val AspIle Pro Gly Thr Ser Val 145 150 155 160 Ala Ile Ser Asn Cys Asp Gly LeuAla Gly Met Ile Lys Ser Asp Asn 165 170 175 Glu Glu Tyr Phe Ile Glu ProLeu Glu Arg Gly Lys Gln Met Glu Glu 180 185 190 Glu Lys Gly Arg Ile HisVal Val Tyr Lys Arg Ser Ala Val Glu Gln 195 200 205 Ala Pro Ile Asp MetSer Lys Asp Phe His Tyr Arg Glu Ser Asp Leu 210 215 220 Glu Gly Leu 2254 720 DNA Homo sapiens 4 gctttgccca gtagttggaa agtgaactcg actcgtgatggttctcctgt cactttggtt 60 gatagcagcc gctctggtag aggttaggac ttcagctgatggacaagctg gtaatgaaga 120 aatggtgcaa atagatttac caataaagag atatagagagtatgagctgg tgactccagt 180 cagcacaaat ctagaaggac gctatctctc ccatactctttctgcgagtc acaaaaagag 240 gtcagcgagg gacgtgtctt ccaaccctga gcagttgttctttaacatca cggcatttgg 300 aaaagatttt catctgcgac taaagcccaa cactcaactagtagctcctg gggctgttgt 360 ggagtggcat gagacatctc tggtgcctgg gaatataaccgatcccatta acaaccatca 420 accaggaagt gctacgtata gaatccggaa aacagagcctttgcagacta actgtgctta 480 tgttggtgac atcgtggaca ttccaggaac ctctgttgccatcagcaact gtgatggtct 540 ggctggaatg ataaaaagtg ataatgaaga gtatttcattgaacccttgg aaagaggtaa 600 acagatggag gaagaaaaag gaaggattca tgttgtctacaagagatcag ctgtagaaca 660 ggctcccata gacatgtcca aagacttcca ctacagagagtcggacctgg aaggccttga 720

What is claimed is:
 1. An isolated polynucleotide comprising a nucleicacid sequence encoding an amino acid sequence which is at least 95%identical to SEQ ID NO:
 1. 2. The isolated polynucleotide of claim 1,wherein said amino acid sequence comprises SEQ ID NO:
 1. 3. The isolatedpolynucleotide of claim 1, wherein said nucleic acid sequence encodes ametalloprotease.
 4. The isolated polynucleotide of claim 1, wherein saidpolynucleotide comprises SEQ ID NO:
 2. 5. An isolated polynucleotidewhich hybridizes under stringent conditions to a nucleic acid moleculecomprising SEQ ID NO: 1 or to a sequence which is complementary tonucleotides of SEQ ID NO:
 1. 6. An isolated polynucleotide comprision asequence which is complementary to the protein encoding sequence of thepolynucleotide of claim
 1. 7. An expression vector comprising apolynucleotide of claim
 1. 8. A host cell transformed or transferredwith an expression vector of claim
 7. 9. A method for producing ADAMTS-3protein, said method comprising the steps of (a) culturing a host cellof claim 8 under conditions suitable for expression of an ADAMTS-3protein; and (b) recovering said ADAMTS-3 protein from the host cellculture.
 10. The isolated polynucleotide of claim 1 wherein said aminoacid sequence is at least 97% identical to SEQ ID NO:
 1. 11. Theisolated polynucleotide of claim 1 wherein said amino acid sequencewhich is at least 99% identical to SEQ ID NO:
 1. 12. A substantiallypurfied procollagen peptidase comprised of a peptide selected from thegroup consisting of functional equivalents of ADAMTS-3, variants ofADAMTS-3, analogs of ADAMTS-3 and fragments of ADAMTS-3.
 13. Thesubstantially purified procollagen peptidase of claim 12, wherein saidprocollagen peptidase is a procollagen I/II N-propeptidase.
 14. Thesubstantially purified procollagen peptidase of claim 12 wherein saidpeptidase is a human peptidase.
 15. The substantially purifiedprocollagen peptidase of claim 12 wherein said peptidase is a bovinepeptidase.
 16. The substantially purified procollagen peptidase of claim12 wherein said procollagen peptadse is capable of processingprocollagens.
 17. An isolated polynucleotide comprising a nucleic acidsequence which is at least 95% identical to SEQ ID NO:
 2. 18. Theisolated polynucleotide of claim 17, said polynucleotide encoding for aprotein which processes procollagen II.
 19. An isolated polynucleotidecomprising a nucleic acid sequence which is at least 95% identical toSEQ ID NO:
 4. 20. The isolated polynucleotide of claim 19, saidpolynucleotide encoding for a protein which processes procollagen II.